In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which primary collagen defects cause skeletal fragility and other significant connective tissue symptoms and then to apply the knowledge gained from our studies to the treatment of children with these conditions. An understanding of the interactions of mutant collagen molecules with the normal collagenous and non-collagenous components of extracellular matrix will enhance our understanding of normal bone function and may yield insights into the more common forms of osteoporosis. We recently focused on the development of a non-lethal animal model for OI with a classical collagen mutation. This non-lethal knockin mouse, the Brtl mouse, with a glycine substitution mutation in the alpha1(I) chain, is an excellent model for pharmacological treatment trials, for approaches to gene therapy suitable for dominant disorders, and for investigations of the skeletal matrix of OI. Our clinical studies involve children with types III and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for treatment.
The OI/EDS region of the alpha1(I) collagen chain
Cabral, Letocha, Marini; in collaboration with Colige, Leikin
In collaboration with Sergey Leikin and Alain Colige, we have succeeded in associating the distinct OI/EDS phenotype with mutations in type I collagen, in identifying the structural basis in collagen for the phenotype, and in delineating the mechanism by which the mutations exert their effect. Our work has focused on seven probands with combined OI/EDS (Ehlers-Danlos syndrome) with type IV or III OI, plus severe laxity of large and small joints and early progressive scoliosis. We determined that the seven probands had mutations in the first 90 residues of the helical region of the alpha1(I) chain and that the thermal stability of collagen differed from that of procollagen in each of these mutations, in contrast to normal procollagen and collagen, which have a single identical melting peak. This is also in contrast to collagen mutations beyond the first 90 amino acids; their differential scanning calorimetry tracings usually show both normal and lower stability peaks, even though the melting curves of procollagen and collagen are identical for each mutation. The assays define a distinct high-stability folding region at the end of the helical region of type I collagen that is disrupted by the presence of the OI/EDS mutations. The unfolding of the anchor region caused by the mutations extends into the N-propeptide. The resulting pN-collagen is incorporated into matrix deposited by cultured fibroblasts, with pN-alpha1(I) collagen prominently present in the newly incorporated and immaturely cross-linked fractions. Electron microscopy of dermal fibrils of six patients revealed that fibril diameters of all six were significantly smaller than those of matched controls, as is seen in EDS VII A and B owing to the absence of the N-proteinase cleavage site from the alpha1(I) or alpha2(I) chain, respectively. The assays also define a folding region of alpha1(I) in which mutations cause a distinct OI/ED phenotype by altering the triple-helical structure and secondary structure of the N-proteinase cleavage site. The data provide a mechanism for the EDS symptoms while the helical changes per se are responsible for bone fragility. The abnormal fibrils may cause laxity of joints and paraspinal ligaments directly through reduced resistance to shearing forces or indirectly by altering interactions between collagen and other matrix components in the overlap zone of the D-periods.
Cabral WA, Makareeva E, Colige A, Letocha AD, Ty JM, Yeowell HN, Pals G, Leikin S, Marini JC. Mutations near amino end of alpha1(I) collagen cause combined OI/EDS by interference with N-propeptide processing. J Biol Chem 2005;280:19259-19269.
Kuznetsova NV, Forlino A, Cabral WA, Marini JC, Leikin S. Structure, stability and interactions of type I collagen with Gly 349 Cys substitution in alpha1(I) chain in a murine osteogenesis imperfecta model. Matrix Biol 2004;23:101-112.
Type I collagen C–propeptide mutations
Marini, Barnes, Ashok
Mutations in the C-propeptide of type I collagen have been found in a small number of patients with OI. The phenotype of these patients ranges from lethal to moderately severe. The mutations are of special interest because they are located in a region that is cleaved from procollagen before collagen fibril assembly. Therefore, the mutations per se are not expected to be present in collagen fibrils in tissues, suggesting that the pathophysiological mechanism of the mutations must differ from mutations in the helical region of the alpha chains, which are incorporated into matrix and exert a dominant-negative effect.
We identified five novel C-propeptide mutations at conserved residues in the collagen of children with types III and IV OI. All mutations delayed incorporation of alpha1 chains into heterotrimers, with delay ranging from two to six times the chain incorporation time of normal control collagen. A pericellular processing assay suggests that a delay occurs in C-propeptide removal from secreted collagens containing the mutations. Mutant collagens are incorporated into fibroblast matrix in culture and form mature cross-links.
Using immunofluorescence assays, we compared the intracellular interaction of the mutant procollagen molecules with endoplasmic reticulum (ER) chaperones in OI fibroblasts with normal controls and cells with a C-terminal helical mutation. Our results demonstrate a clear correlation between the presence and type of mutation with the subcellular localization pattern of procollagen. Normal procollagen and procollagens with mutations in the carboxyl end of the helical domain display a distinct reticular pattern of ER localization while the procollagens with C-propeptide mutations exhibit diffuse ER localization. The diffuse pattern of procollagen staining displays an almost complete overlap with the immunofluorescence pattern of the ER chaperones Hsp-47 and protein disulfide isomerase and minimal overlap with the ER membrane–associated chaperone calnexin. In contrast, the reticular pattern of procollagen fluorescence observed in normal and helical mutation–bearing fibroblasts shows poor overlap with both Hsp-47 and PDI but highly significant overlap with calnexin. Thus, procollagens with mutations near the carboxyl end of the helix and C-propeptide mutations display different intracellular behavior. The location of the mutation along the procollagen chains directs the nature of ER-chaperone interactions.
Alendronate treatment of Brtl mouse
Marini, Uveges; in collaboration with Goldstein, Gronowicz
Bisphosphonate drugs are widely administered to children with OI, but their effects on OI bone containing abnormal type I collagen have not been directly examined. The Brtl mouse model for type IV OI has a glycine substitution (G349C) knocked into one COL1A1 allele. We treated Brtl and wild-type offspring of Brtl x CD-1 matings from 2 to 14 weeks of age with alendronate (0.219 mg/kg/wk, gift of Merck) or saline placebo. Brtl mouse weight and femur length were significantly smaller than in wild type and unchanged by alendronate.
Whole-bone density of femurs and lumbar vertebrae, measured using a Lunar Piximus, were significantly elevated in both treated Brtl and wild type; treated Brtl samples attained average untreated wild-type bone mineral density (BMD). Micro computer tomographic data suggest that the differences in Brtl are attributable to increases in bone volume rather than to mineralization. Owing to an increase in trabecular number, distal femoral bone volume per total volume doubled with treatment in both Brtl and wild type. Diaphyseal cortical thickness increased by periosteal bone deposition in Brtl and wild-type femurs. In treated Brtl femurs, overall geometry reshaped to a more rounded structure, similar to that of untreated wild type. We tested the mechanical properties of femurs in four-point bending. Alendronate treatment increased femoral stiffness and decreased pre-yield displacement in wild type, indicating that the treatment is not benign for normal bone. Stiffness, pre-yield displacement, and yield load did not change in treated Brtl femora. For both genotypes, femurs fractured at an increased ultimate load.
Unfortunately, alendronate had a negative impact on several aspects of bone quality. First, it decreased the predicted material strength and modulus of Brtl and wild-type bone. Second, it did not improve the brittleness (post-yield displacement) of treated Brtl femurs; in fact, post-yield displacement decreased further in treated Brtl compared with untreated wild type. After they reached the yield point, Brtl femurs fractured with similar additional load and deformation as if untreated. Third, the metaphyses of treated Brtl femurs exhibited an increase in remnants of mineralized cartilage. Attributable to the presence of mineralized cartilage in the bone, the matrix discontinuities may increase the risk of fracture initiation and account for some of the observed increase in BMD. Fourth, treatment had a detrimental effect on bone cells. After 12 weeks of alendronate treatment, bone formation rate per bone surface (BFR/BS), mineral apposition rate (MAR), and mineralized surface per bone surface (MS/BS) declined to less than 25 percent of pretreatment values in Brtl and wild type. At that time, the percent of osteoblast surface was lower in both genotypes while the percent of osteoclast surface was stable. In addition, the morphology of the Brtl osteoblasts changed from the plump cuboidal osteoblasts seen in untreated femurs to an intermediate morphology, thus supporting a toxic effect on the cells. Our interpretation is that alendronate treatment of Brtl improves bone geometry and increases loading before fracture but decreases predicted bone material quality and alters osteoblast surface and morphology. The data suggest that a limited treatment duration may be optimal for obtaining improved bone geometry and minimizing the detrimental effects of extended treatment on bone quality.
Pamidronate treatment of children with types III and IV OI
Letocha, Marini; in collaboration with Gerber, Paul
Uncontrolled trials of bisphosphonates in OI children report increased vertebral bone density and height, improved strength and functional level, and decreased fractures and bone pain. We undertook a randomized controlled trial of pamidronate in children with Types III and IV OI. The first study year was controlled; children in the treatment group received pamidronate (10 mg/m2/day for 3 days every 3 months). Children in both treatment and control groups underwent quarterly rehabilitation and physical therapy assessments, including measurements of function, strength, and pain. Children in the treatment group received pamidronate for an additional 6 to 21 months. All patients had L1-L4 DEXA, spine qCT, spine radiographs, musculoskeletal and functional testing. In the controlled phase, treated patients experienced a significantly higher vertebral BMD z-score than controls. They also had significantly greater L1-L4 mid-vertebral height and total vertebral area than controls. The treatment group did not, however, experience fewer long-bone fractures. In the extended treatment phase, patients’ DEXA z-scores, vertebral heights, and areas did not increase significantly beyond the 12-month value.
In the context of maximized physical rehabilitation, we did not see an additional functional effect from bisphosphonate treatment. In contrast to reports from uncontrolled trials, we found no significant changes in ambulation level, lower-extremity strength, or pain in OI children treated with pamidronate. We used the 10-point Brief Assessment of Motor Function (BAMF) to assess motor skills related to ambulation. In the treatment group BAMF at initiation was 6.1±1.8 versus 6.7±1.9 at 12 months. In the control group BAMF at initiation was 6.6±2 versus 7.02±1.31 at 12 months . We assessed manual muscle testing as the sum (total points 110) of abdominal, straight-leg raise, hip abduction, extension, and flexion and quadriceps strength. Lower-extremity muscle strength did not change. The treatment group score was 67.1±20.9 at baseline versus 68.6 ± 19.7 at 12 months; the control group score was 74.2±21.3 at initiation versus 74.6±14.3 at 12 months. We observed no significant decrease in pain on a four-point scale. Some patients reported increased endurance or decreased back pain, but most reported no perceptible changes. The previously reported changes in these parameters appear to have been placebo effects in the uncontrolled trials.
Interestingly, the treatment group exhibited considerable variability of response. Some children had a robust response in all measurements, whereas others had increased bone density but not increased area or height. Changes in DEXA z-scores ranged from less than one standard deviation (SD) to more than three SDs. Such variability of response has not been previously reported and is presumably related to the differences in bone matrix caused by the underlying collagen mutations.
Kozloff KM, Carden A, Berwitz C, Forlino A, Uveges TE, Morris MD, Marini JC, Goldstein SA. Brittle IV mouse model for osteogenesis imperfecta IV demonstrates post pubertal adaptations to improve whole bone strength. J Bone Miner Res 2004;19:614-622.
Letocha AD, Cintas HL, Troendle JF, Reynolds JC, Cann CE, Chernoff EJ, Hill SC, Gerber LH, Marini JC. Controlled trial of pamidronate in children with types III and IV osteogenesis imperfecta confirms vertebral gains but not short-term functional improvement. J Bone Miner Res 2005;20:977-986.
Marini JC, Chernoff E, Letocha AD. Osteogenesis imperfecta. In: Allanson J, Cassidy S, eds. Clinical Management of Common Genetic Syndromes, 2nd edition. New York: Wiley and Sons, 2004;407-420.
Walker LC, Overstreet MA, Willing M, Marini JC, Cabral WA, Pals G, Bristow P, Atsawasuwan M, Yamauchi M, Yeowell HN. Heterogeneous basis of the type VIB form of Ehlers-Danlos Syndrome (EDS VIB) that is unrelated to decreased collagen lysyl hydroxylation. Am J Med Genet 2004;131A:155-162.
11now at the University of Pavia, Pavia, Italy
COLLABORATORS
Alain Colige, PhD, Université de Liège, Liège, Belgium
Lynn Gerber, MD, Rehabilitation Medicine, Warren G. Magnuson Clinical Center, Bethesda, MD
Steven Goldstein, PhD, University of Michigan, Ann Arbor, MI
Gloria Gronowicz, PhD, University of Connecticut Health Center, Farmington, CT
Sergey Leikin, PhD, Section on Physical Biochemistry, NICHD, Bethesda, MD
Scott Paul, MD, Rehabilitation Medicine, Warren G. Magnuson Clinical Center, Bethesda, MD
For further information, contact marinij@mail.nih.gov.